Project Summary Lymphocyte migration and recirculation has long been thought to play a critical role in homeostatic lymphocyte survival. This is certainly the case for T cells where migration within the T cell zone and interactions with fibroblastic reticular cells enables access to interleukin-7, a cytokine that together with tonic signals from the T cell receptor promotes long-term T cell maintenance. Similarly, B cell homeostasis is controlled by a combination of tonic signaling from the B cell receptor (BCR) and B cell Activating Factor (BAFF) receptor. However, neither tonic BCR signaling nor BAFF receptor signaling are anatomically restricted (e.g. secondary lymphoid organs). Therefore, the generalized notion that B cell survival is linked to their intrinsic ability to migrate within and between lymphoid organs is not easily compatible with the current knowledge of B lymphocyte homeostasis. In recent and preliminary studies we tested the impact of G alpha inhibitory (Gi) protein-coupled chemoattractant receptor signaling in B cell homeostasis by examining the B cell compartment of mice in which Pertussis toxin (PTX) expression is enforced in B cells. In this grant we provide evidence showing that PTX+ B cells have severe defects in transitional B cell differentiation into mature B cells in the spleen, and defects in mature B cell survival. Furthermore, we show that BCR signaling is significantly increased in PTX+ B cells, which results in a dramatic increase in DNASE1L3 expression and nuclear localization. PTX+ B cells have increased DNA damage, and we hypothesize that the BCR-dependent increase in DNASE1L3 contributes to the defects seen in transitional B cell differentiation and mature B cell homeostasis. In this grant we propose to examine the mechanism(s) whereby Gi protein signaling attenuates tonic BCR signaling, and to measure the impact of DNASE1L3 in removal of aberrant cells from the B cell compartment. In specific aim 1 we will test whether reducing BCR signaling from PTX+ B cells rescues the B cell differentiation and survival defects. We will further characterize protein-protein interactions established by GTP-Gi proteins by mass spectrometry analyses of proteins co-immunoprecipitated with Gi in PTX+ and PTX- B cells. In specific aim 2 we will test the impact of Dnase1l3 genetic deficiency in PTX+ B cell transitional differentiation and mature B cell survival. We will also quantify the extent to which DNASE1L3 causes DNA damage in PTX+ B cells. Combined, these studies will not only provide new insights into the fundamentals of B cell differentiation and homeostatic survival, but may also provide insight into new molecular targets for manipulating B cells in vivo for the treatment of B cell-mediated autoimmune diseases, immunodeficiency, and B cell malignancies.